Accurate replication of the genome and continuous surveillance of its integrity are essential for cell survival and avoidance of diseases such as cancer and premature aging. The replication stress response (RSR) recognizes challenges to DNA replication from insults such as ionizing radiation and coordinates diverse DNA repair and cell cycle checkpoint pathways necessary to maintain genome integrity. However, the mechanisms mediating many RSR activities and how their dysregulation leads to carcinogenesis are poorly understood. Thus, the overarching goal of this proposal is to determine the critical processes by which cells respond to replication stress and apply this knowledge for cancer prevention and treatment. Preliminary data identify SIRT2, a sirtuin family deacetylase, as a novel regulator of the RSR. Prior work has implicated sirtuins in both aging and tumor suppression. Specifically, mice deficient in Sirt2 develop breast and liver cancers, and SIRT2 expression is decreased in human breast and liver cancers, suggesting that SIRT2 is a legitimate tumor suppressor. Recent data from the lab show that SIRT2 deacetylates CDK9 at a conserved lysine residue to promote recovery from replication arrest. Mass spectrometry approaches also identified a number of additional RSR proteins that interact with and are deacetylated by SIRT2. These findings imply a critical role for SIRT2 in the RSR that may provide an underlying mechanism by which aging and carcinogenesis are connected; however, the precise functions of SIRT2 in the RSR are not clear. As such, the overall objective of this proposal is to determine the mechanisms by which SIRT2 functions in the RSR to maintain genome integrity. The hypothesis being tested is that SIRT2 maintains genome integrity, at least in part, by directing the RSR through deacetylation of CDK9 and other key substrates. The following specific aims are proposed: 1. Determine and mechanistically dissect the specific role that SIRT2 plays in directing the RSR, 2. Determine the functional significance of SIRT2-mediated deacetylation of CDK9 in the RSR, 3. Identify SIRT2 substrates that function in the RSR. This proposal is conceptually innovative in that it proposes a novel mechanism for regulation of the RSR that involves SIRT2 as an orchestrator of the RSR through deacetylation of critical RSR proteins, establishing a novel paradigm by which RSR proteins are regulated by deacetylation and establishing a novel role for SIRT2, and more generally for sirtuins, in the regulation of the RSR. Completion of these aims will provide new insights into how SIRT2 maintains genome integrity and prevents cancer, elucidate the significance of the SIRT2 acetylome in regulating the RSR, and improve our understanding of the link between aging and carcinogenesis. Recently, the NCI has proposed a new research emphasis to design rigorous and innovative research strategies to solve specific problems and paradoxes in cancer research identified as the NCI's Provocative Questions. This research proposal addresses one such question What mechanisms of aging, beyond the accumulation of mutations, promote or protect against cancer development?